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nature structural biology • volume 7 number 2 • february 2000 127

Structural origins of theselectivity of thetrifunctional oxygenaseclavaminic acid synthaseZhihong Zhang1,2, Jingshan Ren1,3, David K. Stammers3,Jack E. Baldwin2, Karl Harlos3 and Christopher J. Schofield2

1These authors contributed equally to the work. 2The Dyson PerrinsLaboratory and the Oxford Centre for Molecular Sciences, SouthParks Road, Oxford OX1 3QY, UK. 3Structure Biology Division, TheWellcome Trust Centre for Human Genetics and the Oxford Centrefor Molecular Sciences, Roosevelt Drive, Oxford, OX3 7BN, UK.

Clavaminate synthase (CAS), a remarkable Fe(II)/2-oxoglu-tarate oxygenase, catalyzes three separate oxidative reactionsin the biosynthesis of clavulanic acid, a clinically usedinhibitor of serine β-lactamases. The first CAS-catalyzed step(hydroxylation) is separated from the latter two (oxidativecyclization/desaturation) by the action of an amidinohydro-lase. Here, we describe crystal structures of CAS in complexwith Fe(II), 2-oxoglutarate (2OG) and substrates (N-α-acetyl-L-arginine and proclavaminic acid). They revealhow CAS catalyzes formation of the clavam nucleus, viaa process unprecedented in synthetic organic chem-istry, and suggest how it discriminates between sub-strates and controls reaction of its highly reactive ferrylintermediate. The presence of an unpredicted jelly roll β-barrel core in CAS implies divergent evolution withinthe family of 2OG and related oxygenases. Comparisonwith other non-heme oxidases/oxygenases reveals flexi-bility in the position which dioxygen ligates to the iron,in contrast to the analogous heme-using enzymes.

Soon after the introduction of β-lactam compounds asantibiotics, it became apparent that bacteria had devel-oped resistance mechanisms involving β-lactamases1–3,which catalyze the hydrolysis of the β-lactam ring to givebiologically inactive products (Fig. 1a)4. Most clinicallyimportant β-lactamases employ a mechanism involving anucleophilic Ser residue and proceeding via a hydrolytical-ly labile acyl–enzyme intermediate4. In attempts to over-

come the action of β-lactamases, a massive effort in medicinalchemistry has been made to discover β-lactam antibiotics resis-tant to the β-lactamase mediated hydrolysis. Partial successes,such as the use of cephalosporins and carbapenems have beenachieved; however, this strategy has only led to temporary solu-tions. Alternatively, selective and efficient inhibitors of β-lacta-mases have been searched for.

The most important serine β-lactamase inhibitor is a naturalcompound, clavulanic acid, which possesses little antibacterialactivity and is consequently administered in combination withan antibiotic5–7. It inhibits class A serine β-lactamases through acomplex mechanism involving sequential acylation, decarboxy-lation and loss of a four carbon fragment to give stable acyl-enzyme complexes (Fig. 1b)8,9. Despite its small size and the factit contains only two chiral centers, to date there has not been anasymmetric synthesis of optically active clavulanic acid (low-yielding formal syntheses of racemic methyl clavulanate havebeen reported10,11) and instead, it is produced by fermentation ofStreptomyces clavuligerus.

Like the penicillins, clavulanic acid contains a strained bicyclicβ-lactam ring system. Penicillin biosynthesis occurs via theisopenicillin N synthase (IPNS) mediated oxidative cyclizationof L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) toisopenicillin N (Fig. 1c)12–14. IPNS is related, both by sequenceand its requirement for Fe(II) and dioxygen, to the extendedfamily of 2-oxoglutarate (2OG) dependent oxygenases (Fig. 1d).These enzymes catalyze a very wide range of oxidative reactions

Fig. 1 β-Lactam antibiotics (resistance and biosynthesis) and 2-oxoglutarate dioxygenases. a, Mechanisms of action for β-lactamantibiotics via irreversible acylation of transpeptidase enzymes andfor serine β-lactamases4. b, Mechanism for the inhibition of serineβ-lactamases by clavulanic acid8,9. c, The isopenicillin N synthasecatalyzed conversion of L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine(ACV) to isopenicillin N and the oxidative ring expansion of peni-cillin N to deacetoxycephalosporin C (DAOC) as catalyzed by the2OG-dependent oxygenase, DAOCS. d, Stoichiometry of a typicalhydroxylation reaction as catalyzed by a 2OG-dependent dioxyge-nase. Incorporation of an oxygen atom from dioxygen into thehydroxyl group is normally less than stoichiometric becauseexchange with water occurs. e, Comparison of the coordinationchemistry of (i) CAS (ii) DAOCS and (iii) IPNS. The dioxygen bindingsite in IPNS is trans to Asp 216 (ref. 13). In CAS and DAOCS, watermolecules occupy the sites opposite His 279 and His 183, respec-tively. The possibility of rearrangement upon dioxygen bindingcannot be ruled out. It is interesting that there are clear similaritiesbetween the metal binding sites of CAS (and other iron oxygenas-es/oxidases) with zinc-dependent hydrolases including ther-molysin47 and the metal-dependent (zinc) β-lactamases3,4.

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in nature, including steps in the biosynthesis of collagen, carni-tine, plant signaling molecules and many antibiotics15. The oxi-dase IPNS is unusual in that it does not use a 2OG cofactor,instead catalyzing the four-electron oxidation of its tripeptidesubstrate. Crystal structures have been reported for IPNS12,13 andfor the 2OG oxygenase, deacetoxycephalosporin C synthase(DAOCS)16,17, which catalyzes the unusual oxidative ring expan-sion of penicillin N to DAOC (Fig. 1c). The DAOCS structures,the first of a 2OG-dependent oxygenase, revealed how iron and2OG were bound at its active site. However, it has not yet beenpossible to obtain the structure of this enzyme complexed to itspenicillin N substrate, nor have any other structures of 2OG oxy-genases catalyzing more typical reactions been reported.

In contrast to penicillin biosynthesis, the pathway to clavulan-ic acid is convoluted and proceeds via clavaminic acid (A2),which but for the substitution of an amine for an alcohol is theenantiomer of clavulanic acid itself 7 (Fig. 2a). The monocyclicβ-lactam (A1)18,19 is converted in four steps to clavaminic acid(A2), which undergoes a double epimerization via an unknownprocess to give a labile aldehyde (A3), which is reduced in anNADPH-dependent reaction to give clavulanic acid20. A single2OG oxygenase enzyme, clavaminic acid synthase (CAS), plays apivotal role in the pathway catalyzing the construction of the

128 nature structural biology • volume 7 number 2 • february 2000

labile bicyclic clavam ring system21–24. In the firstCAS catalyzed reaction, the monocylic β-lactam(A1) is hydroxylated to give an alcohol (A4) that isneither a substrate nor inhibitor of CAS. Instead,proclavaminate amidinohydrolase (PAH)25 cat-alyzes the hydrolysis of the guanidino side chain ofA4 to give proclavaminic acid (A5), which is oxida-tively cyclized by CAS to give dihydroclavaminicacid (A6). In the final CAS-catalyzed reactiondihydroclavaminic acid (A6) is desaturated toclavaminic acid (A2)22. In S. clavuligerus, CASexists as two functionally identical isozymes (CAS1and CAS2) with a sequence identity of 87% (refs 24,26) and is involved in the biosynthesis ofother clavam antibiotics7. In the present study theCAS1 isozyme was used.

All oxygenase catalyzed reactions involve the ini-tial activation of kinetically inert dioxygen fol-lowed by the generation of a reactive oxidizingintermediate, the reactivity of which must bedirected to ensure product selectivity, for example,

hydroxylation rather than desaturation. The process by which anenzyme controls the reactivity of a highly reactive intermediatehas been referred to as negative catalysis27. With CAS the type ofreaction catalyzed is dependent upon the precise structure of thesubstrate; that is, oxidation of a β-lactam derivative (A1) with aguanidino side chain results in an alcohol (A4), whereas oxida-tion of the corresponding derivative with an amino side chaingives predominantly an alkene7,23. This makes CAS an excellentchoice for structural investigations aimed at understanding thegeneral principles of how the substrate and product selectivitiesof oxygenases are achieved. Note that there is little or no synthet-ic precedence for the range of difficult oxidative chemistry cat-alyzed by CAS, including hydroxylation, oxidative cyclizationand desaturation.

Topology and iron coordinationThe main chain of CAS1 contains a core of ten β-strands (β1, β2,β4–β7, β11–β14), eight of which are folded into a distorted jellyroll topology (β2,β5−β7,β11−β14)(Fig. 3a,b). The jelly roll coreis sandwiched between two largely α-helical regions: an N-ter-minal region (residues 2–118) containing four helices (α1–α4)and an extended insert (residues 177–258) containing α-helices(α5–α7) and β-strands (β8–β10), which links the fourth (β7)

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Fig. 2 The role of CAS in clavulanic acid biosynthesis. a, Biosynthetic pathway to clavulanic acid. Each CAS cat-alyzed step is coupled to the conversion of 2OG anddioxygen to succinate and CO2. The oxygen introducedand hydrogen removed during the CAS catalyzed reac-tions are in red. BLS: β-lactam synthetase; PAH: pro-clavaminate amidinohydrolase. b, Mechanism for theCAS catalytic cycle. R = CH2CH2CO2H. See text for discus-sion. c,d, Relationships between the ferryl intermediateand two substrates in the CAS catalyzed hydroxylationand cyclization processes. The involvement of proteinside chains in the cyclization and desaturation processescannot be ruled out entirely. Due to the instability of A6,it has not yet been possible to obtain a structure ofCAS–A6 complex. The available structures suggest thebicyclic clavam ring of dihydroclavaminic acid (A6) allowsit to bind in such a manner as to direct the two C–H bondsthat must be oxidized towards the reactive iron centerwithout the need for invoking a very different bindingsite for its basic side chain compared to those of NAA orproclavaminic acid (A5).


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and fifth (β11) strands of the jelly roll core. Three flexible loops,as indicated by their crystallographic temperature (B) factors,border the active site (Fig. 3d). Two of these loops (residues200–215 and 231–241) come from the extended insert and pro-vide acidic residues (Asp 202, Asp 233) involved in binding thebasic side chains of the substrates (Fig. 3d). Residues from theformer loop prevent the C-terminus from approaching the activesite. The third flexible loop (residues 124–140) forms a lid overthe bound 2OG in the active site.

In the CAS–Fe(II) complex the iron is ligated in an almostoctahedral geometry by the side chains of His 144, Glu 146, His279 and water molecules. The side chain carboxylate of Glu 146binds the iron in a monodentate fashion with its other oxygenpositioned to form a hydrogen bond with the hydroxyl group ofTyr 299. The presence of a glutamyl rather an aspartyl iron ligandis unprecedented in 2OG oxygenases and IPNS, but conservesthe two histidines-one carboxylate motif found in many non-heme iron oxygenases28,29. This motif, which projects from thepresumably rigid β-barrel core of the 2OG oxygenases, may ful-fill a role similar to that of the porphyrin ring in cytochromeP450 enzymes to provide a stable platform to ligate the iron andthe subsequently formed reactive intermediates. An aspartate


may be generally preferred as an iron ligand in 2OG oxygenasesbecause its side chain is more rigid than that of a glutamate. Thepresence of a glutamate as an iron ligand in CAS may reflect theneed for flexibility to perform its trifunctional catalytic role.

Sequence analyses of CAS and other 2OG oxygenases12,15,26

have revealed little overall similarity to the DAOCS/IPNS sub-family, leading to the speculation that convergent evolution to acommon mechanism/active site chemistry occurred within thewider family of 2OG and related oxygenases26. The presence of ajelly roll topology in CAS like those in IPNS12 and DAOCS16, inwhich certain analogous motifs are located on analogous strandsof the core, implies a divergent evolutionary relationshipbetween CAS and other members of the extended family of 2OGand related enzymes. The conserved motifs include the ironbinding residues on or close to the second and seventh jelly rollstrands (β5 and β13 of CAS respectively), an arginine binding tothe 5-carboxylate of 2OG at the beginning of the eighth jelly rollstrand (β14 of CAS), and the residues constituting the primesubstrate binding/active site (Fig. 3a). This analysis also suggeststhat all of the 2OG oxygenases, including mammalian enzymessuch as prolyl-4-hydroxylase28, may contain a jelly roll β-barrelcore. Primary sequence analyses with CAS might have been mis-

Fig. 3 The structure of CAS. a, Stereoview of the CAS–Fe(II)–2OG complex structure showing the entrance to the active site (β-strands in green, iron inpink). 2OG and some of the substrate binding residues discussed in the text are in yellow. b, Alternative view showing the jelly roll β-strand core (in green)of CAS that is conserved in many 2OG and related oxygenases. The extended insert linking the fourth (β7) and fifth (β11) strands of the jelly roll core is inpink and the N-terminal region is in light purple. c, Stereoview of the electron density map of the active site of CAS bound to iron (II) and 2OG. The dis-tances (calculated using the described refinement procedure45) from the iron to its ligands are His 144: 2.15 Å; Glu 146: 2.06 Å; His 279: 2.12 Å; 2-oxo groupof 2OG: 2.21 Å; carboxylate oxygen of 2OG: 2.01 Å; water: 2.21 Å. The 2|Fo| - |Fc| electron density map is contoured at 2.5 σ. d, The structure colored accord-ing to crystallographic temperature factors with blue as the lowest and red as the highest value. Selected iron and substrate binding residues are labeled.Note the three loops that border the active site in high B-factor regions. Panel (a,b) was created using MOLSCRIPT48, (c,d) using BOBSCRIPT49 and the pic-tures were rendered with RASTER3D50.

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led by the displacement of the β-barrel core within the primarysequence and by alterations to the structure around the core,including the extended loop linking the fourth and fifth strands(β7 and β11 in CAS).

2-oxoglutarate and dioxygen bindingKinetic studies of 2OG oxygenases indicate an ordered sequen-tial mechanism, with binding of 2OG followed by that of primesubstrate, then dioxygen. Product release occurs in the follow-ing order: bicarbonate (or CO2), succinate and prime product30.The order of release of the latter two is dependent on their rela-tive concentrations30. Electron transfer from the iron to thedioxygen may simultaneously permit formation of an ironbound superoxide or peroxide species and activate keto-groupof 2OG for nucleophilic attack resulting in formation of a per-oxo species, which then collapses to give the ferryl [Fe(IV)=O]species, responsible for substrate oxidation (Fig. 2b)31. Closelyrelated ferryl species are proposed to be involved in the catalysisby porphyrin-dependent oxygenases [Fe(V)=O] and di-ironoxygenases, such as methane monooxygenase [Fe(III)–O–Fe(V)=O], which also catalyze the oxidation of unactivatedC–H bonds32. The precise orientation of the substrates withrespect to the highly reactive ferryl species is vital to ensure thatthe correct bonds are oxidized. The structures of CAS com-plexed with substrates provide the first insights into the spatialrelationships between the reactive oxidizing species and the sub-strates in 2OG oxygenase catalysis.

In the CAS–Fe(II)–2OG complex (Fig. 3c) two ligating watermolecules are replaced by the 2-oxo acid group of 2OG thatbinds in a bidentate manner, supporting spectroscopic stud-ies33,34. The high B-factors observed for residues in the loop

(124–140) forming the lid over the 2OG binding siteis consistent with the hypothesis that the lid movesto facilitate binding of the cofactor. The 2-oxo groupis ligated trans to Glu 146 and the 1-carboxylateopposite to His 144. A water molecule occupies theposition opposite His 279. The 5-carboxylate of2OG is in position to form an electrostatic interac-tion with the side chain of Arg 293, which is locatedat the beginning of the last strand of the jelly rollcore (Figs 3, 4). In DAOCS, the 2OG 5-carboxylate isbound by an RXS motif which is conserved in manyof the 2-OG oxygenases15,16. This motif also binds tothe valine carboxylate of ACV in the IPNS–Fe(II)–ACV structure13. The motif is not present in CAS,where the side chain of Leu 295 does point towardsthe 5-carboxylate of 2OG in the active site but can-not form electrostatic or hydrogen bonding interac-

tions. Instead the hydroxyl of Thr 172 (located on the fourthjelly roll strand) is in position to form a hydrogen bond with the2OG 5-carboxylate (Fig. 4a,b). The conservation of both guani-dino and hydroxyl side chains in binding the 5-carboxylate of2OG in both the CAS–Fe(II)–2OG and DAOCS–Fe(II)–2OGstructures suggests a mechanistic importance for these func-tional groups in 2OG binding.

It seems reasonable to suppose that dioxygen binds to the ironatom at the active site of CAS in the position occupied by thewater molecule trans to His 279 (Fig. 3c). If subsequent reactionof dioxygen occurs in this position, the substrate structures sug-gest that a subsequently formed ferryl species would be correctlyoriented to effect oxidation of the requisite C–H bonds.However, it is notable that the relative positions of the metal lig-ating water molecule and the 2OG 1-carboxylate in the CASstructures are reversed relative to those observed in theDAOCS–Fe(II)–2OG16 crystal structure (Fig. 1e). This impliesone or more of the following: (i) dioxygen binding and reactionoccurs at different relative positions in CAS and DAOCS; (ii) oneof the crystal structures, most likely that of DAOCS since it wassolved in the absence of substrate, does not reflect the solutionsituation; or (iii) the different binding properties of 2OG in CASand DAOCS reflect different points in the catalytic cycle. If thelatter is correct it is possible that rearrangement of the DAOCScomplex occurs upon substrate binding. Alternatively, the CAScomplex could rearrange such that dioxygen binds and reacts inthe position trans to His 144, consistent with the DAOCS struc-ture. Subsequent to decarboxylation a five-coordinate ferrylspecies may rearrange from trans to His 144 to trans to His 279.The formation of a five-coordinate intermediate could providean explanation for the observation that during the hydroxylation

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Fig. 4 Active site of CAS. a, Stereoview of the active site of theCAS–Fe(II)–2OG–NAA complex. The 3-pro-R C–H bond of N-α-L-acetyl arginine that is oxidized is in cyan and projectstowards the water ligated to the iron/dioxygen binding posi-tion. The apparent distances from the iron to its ligands areHis 144: 2.14 Å; Glu 146: 2.07 Å; His 279: 2.12 Å; 2-oxo group of2OG: 2.15 Å; carboxylate oxygen of 2OG: 1.94 Å; Wat 2.35 Å.b, Stereoview of the active site of the CAS–Fe(II)–2OG–A5complex. The β-lactam 3-pro-S C–H bond of A5 that is oxidizedis in cyan. The apparent distances from the iron to its ligandsare His 144 Å: 2.25 Å; Glu 146: 2.16 Å; His 279: 2.28 Å; 2-oxogroup of 2OG: 2.16 Å; carboxylate oxygen of 2OG: 2.03 Å.There is insufficient density in the position opposite His 279 tomeasure a reliable bond distance. This figure was createdusing MOLSCRIPT48.



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process, incorporation of oxygen atoms from either dioxygen orwater into the product is observed35–37. Exchange of oxygenderived from dioxygen to that from water may occur in a processinvolving a reaction of water with the five-coordinate ferrylspecies or the subsequently formed Fe(III)–OH intermediate.

Substrate structuresStructures were obtained for CAS–Fe(II)–2OG crystals soakedwith N-α-L-acetylarginine (NAA), which is a stable analog forthe monocylic β-lactam substrate (A1) in the CAS hydroxylationreaction37, and with proclavaminic acid (A5) (Fig. 4). A compar-ison of these two structures suggests how hydrolysis of the sidechain of the guanidino monocyclic β-lactam (A1) to give theamino side chain of proclavaminic acid (A5) could result in theevolution of a second CAS catalyzed reaction. Both NAA andproclavaminic acid (A5) are located in the active site in the samegeneral manner and are less enclosed than 2OG and Fe, consis-tent with initial binding of the 2OG during catalysis. Both the β-lactam of proclavaminic acid (A5) and the acetyl group ofNAA are located in a shallow pocket defined in part by the sidechains of Leu 114, Met 147, Tyr 149, Pro 154, Asp 202, Phe 209and Tyr 299. The pocket is sufficiently large to accommodate theextra methylene of a γ- rather than β-lactam thus rationalizingthe conversion of γ-lactam analogs of proclavaminic acid (A5)and dihydroclavaminic acid (A6)38. Differences in the way thesubstrate side chains are bound change the orientations of thesubstrates with respect to the ferryl [Fe(IV)=O] intermediateand thus the type of oxidative reaction catalyzed (Fig. 2c,d).

In the CAS–Fe(II)–2OG–NAA structure the guanidino groupof NAA is in position to form a planar electrostatic interaction


with the side chain of Asp 233, a hydrogen bond via a water mol-ecule with the backbone carbonyl of Asp 142 and an electrostaticinteraction with Asp 202 (Fig. 4a). Asp 235 is also close to theguanidino binding pocket, but in this structure points away fromthe substrate guanidino group. The carboxylate of NAA is inposition to bind to a water molecule, Arg 297 (located on the lastjelly roll strand) and the backbone NH of Ser 134. The sidechains of Arg 115 and Tyr 299 also point toward the carboxylateof this substrate, but are too distant to form direct interactions,although their involvement in binding via intervening watermolecules or a different solution structure cannot be ruled out.

Binding of NAA in this manner projects its 3-pro-R C–Hbond, which is oxidized, toward the water ligated to theiron/dioxygen binding position, reinforcing the proposal of fer-ryl formation occurring opposite to His 279 (Figs 2d, 4a,b). Twoshort channels, each containing two water molecules, extendtoward this site on the iron, providing a means of entry and exitfor dioxygen/water molecules. Spectroscopic studies indicatethat upon binding of the monocyclic β-lactam to theCAS–Fe(II)–2OG complex a change from a six-coordinate octa-hedral to a five-coordinate square pyramidal geometryoccurs33,34. While a water molecule is still ligated to the iron inthe CAS–Fe(II)–2OG–NAA crystal structure, the apparent bonddistance between the iron and the water increases upon substratebinding (from 2.2 to 2.35 Å). If the hydroxylated product of thefirst CAS catalyzed reaction binds in the same manner as NAA,its hydroxyl group may prevent dioxygen binding, thus suggest-ing why it is not a substrate for CAS.

In the CAS–Fe(II)–2OG–A5 structure, the carboxylate of thesubstrate is also in position to make an electrostatic interaction

Table 1 Data collection, phasing and refinement statistics

Data set Native 1 Native 2 Baker’s1 TML2 CAS1–Fe(II) CAS1–Fe(II) CAS1–Fe(II)–2OG –2OG–NAA –2OG–A5

Resolution (Å) 30–1.63 30–2.49 30–2.50 30–2.50 20–1.08 30–1.40 30–2.10 (outer shell) (1.69–1.63) (2.58–2.49) (2.59–2.50) (2.59–2.50) (1.10–1.08) (1.42–1.40) (2.18–2.10)

Observations 106,073 49,112 44,474 49,650 802,154 181,330 52,595Unique reflections3 35,608 11,448 19,844 20,614 126,607 59,325 18,084Completeness 91.4 98.0 93.7 96.8 92.8 93.9 95.8

(outer shell) (%) (74.6) (89.0) (84.0) (93.9) (57.2) (60.7) (95.0)Rmerge (%)4 4.8 9.0 9.8 7.3 10.8 4.9 9.6I/σ(I) (outer shell) 27.4 (12.2) 21.0 (8.6) 11.7 (3.5) 17.4 (7.3) 16.6 (0.95) 13.6 (1.5) 8.1 (1.6)Isomorphous difference

to Native 2 (%)5 24.3 19.7Number of sites 4 9Phasing power 1.29 / 1.50 / 1.68 / 2.21 /

(acentric / centric / anomalous)6 1.02 1.28RefinementNumber of atoms7 2,481 / 376 2,481 / 446 2,439 / 508 2,435 / 178Rworking / Rfree

8 0.180 / 0.228 0.135 / 0.166 0.174 / 0.205 0.219 / 0.269R.m.s. deviations9

Bond lengths (Å) 0.015 0.016 0.016 0.006Bond angles (°) 2.0 2.7 1.9 1.4

11,4-Diacetoxy mercuri-2,3-dimethoxybutane2Trimethyl-lead acetate.3Anomalous pairs of the derivative data sets were treated as separate reflections.4Rmerge = Σj Σh |Ij,h - <I h>| / Σj Σh <Ih> × 100, where Ij,h are intensities of symmetry redundant reflections and <Ih> = mean intensity of reflection.5Isomorphous difference is defined as Σ ||FPH| - |FP|| / Σ |FP|, where FPH and FP refer to derivative and native structure factors, respectively.6Phasing power is defined in ref. 43 as <[|Fh(calc)| / phase-integrated lack of closure]>.7Protein atoms / water, glycerol, sulphate ion and substrate atoms8R-factor = Σh ||Fo|h - |Fc|h| / Σh |Fo|h where the sum is over the unique reflections, h. For each data set 5% of reflections were randomely selected and notused in refinement. Rfree is the R-factor calculated with these reflections, Rworking is the R-factor calculated from 95% of reflections used in refinement. 9Root mean square deviation from ideality


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with Arg 297, but is slightly closer to Arg 115 than in theCAS–Fe(II)–2OG–NAA structure. The guanidino group ofArg 297 is in position to form hydrogen bonds with the hydroxylgroup of proclavaminic acid (A5) and the 2OG carboxylate,through which the latter is ligated to the iron (Fig. 4b). The sidechain of Ser 134 has rotated relative to the NAA structure so thatit is now in position to form a hydrogen bond with the hydroxylof A5. The amine of A5 seems to bind less rigidly than the guani-dino group of NAA, possibly reflecting conformational changesthat occur during the formation of the bicyclic ring system. Theamine of A5 is in position to form hydrogen bonds with thebackbone carbonyls of Leu 132 and, via a water molecule, withthe side chain of Asp 142. It is further away from the side chainsof Asp 233 and Asp 202 (∼ 5.1 and 3.9 Å, respectively), but mayform interactions with these residues via water molecules notapparent in the crystal structure.

The interactions of Arg 297 and Ser 134 with the hydroxylgroup of proclavaminic acid (A5) apparently hold the substrateaway from the iron, possibly creating a pocket into which dioxy-gen can bind. Indeed, in this structure there is very little electrondensity in the position opposite His 279 and the iron is bestdescribed as predominantly five-coordinate. Hydrogen bondingof the substrate hydroxyl to the dioxygen atom ligated to the ironmay serve to facilitate electron transfer and orient the otherdioxygen atom for attack on the 2OG keto group. Subsequentferryl [Fe(IV)=O] formation is followed by its reaction with the4-pro S β-lactam C–H bond of proclavaminic acid (A5) to givean imminium ion and an iron bound hydroxide (Fig. 4b). Theintermediate iron-bound hydroxide may assist in deprotonationof the hydroxyl in the cyclization process. However, the involve-ment of residues from the protein in the cyclization process can-not be ruled out.

ConclusionsThe insights provided by the CAS structures into the mechanismby which nature synthesizes a strained ring system, and whichhas proved elusive for synthetic chemists, will assist in the designof new oxidizing catalysts, both protein and non-protein based.These structures provide the basis for mutagenesis studies ofCAS aimed at producing modified clavams as antibiotics. It willbe particularly interesting to explore modification of the β-lac-tam binding pocket for the synthesis of clavams with penicillin(acetamido derivatives) or carbapenem (hydroxyalkyl deriva-tives) type side chains, to make hybrid β-lactam antibiotics. Suchcompounds, the preparation of which by traditional syntheticmethods has proved intractable, are likely to have very interest-ing antibacterial and β-lactamase inhibitory activities.

MethodsExpression. The genes encoding both CAS isozymes (CAS1 andCAS2) from S. clavuligerus were cloned into expression vectorpET11a via standard PCR techniques39,40. The genes were expressedin Escherichia coli BL21 (DE3) strain and the CAS isozymes purifiedaccording to slightly modified versions of published procedures37,38.

Crystallization. Crystals of recombinant CAS1 were obtained in2 M (NH4)2SO4 buffered with 100 mM Tris using the hanging dropmethod at 21 °C. Macroseeding was used to obtain large crystals.The crystals belong to space group P212121 with unit cell dimensionsof a = 67.08 Å, b = 67.94 Å and c = 68.7 Å (CAS–Fe(II)–2OG at 100 K);

each asymmetric unit contains one CAS1 molecule (Table 1). TheCAS–Fe(II)–2OG complex could be obtained either by soaking theapo-form crystals or by co-crystallization under anaerobic condi-tions. The enzyme–substrate crystal complexes were preparedanaerobically. Aerobically grown crystals were placed in a nitrogenglove box, equilibrated overnight (minimally) and soaked withcofactors, FeSO4 5 mM, 2OG 5 mM, and N-α-L-acetyl arginine100 mM or proclavaminic acid (A5) 100mM for ~24 h. The X-ray datafor Native 2 and heavy atom derivatives were collected using 18 cmMAR image plates, and for the CAS–Fe(II)–2OG–A5 structure, a34.5 cm MAR image plate. The Native 1 and CAS–Fe(II)–2OG datawere collected at Station 9.6, SRS, Daresbury Laboratory, UK, at awavelength 0.87 Å with a 34.5 cm MAR image plate and a Quantum4 CCD detector, respectively. The data for CAS–Fe(II)–2OG–NAAwere recorded on a 30 cm MAR image plate, at Station 9.5 SRS, at awavelength of 0.80 Å. All data collection were carried out at 100 K,except for Native 2 and heavy atom derivatives which were collect-ed at 21 °C. X-ray images were indexed and integrated withDENZO41 and merged with SCALEPACK41.

Structure Determination. The structure of the apo-enzyme wasdetermined by multiple isomorphous replacement (MIR) (Table 1).Two heavy metal derivatives were prepared by soaking experi-ments: Baker’s dimercurial (5 mM for 70 h) and trimethyl-leadacetate (40 mM for 88 h). Heavy metal binding sites were deter-mined by Patterson and difference Fourier methods. Mlphare42 andSharp43 were used to refine occupancies and calculate phases. Thephases calculated from Sharp43 were further improved by densitymodification using DM44. The resulting electron density map wassufficiently clear to build an initial model of the structure. Structurerefinement was performed using CNS45. The initial model, based onthe MIR map, was first refined against the Native 2 data set to 2.49Å resolution. One round of refinement, which included positional,simulated annealing and individual B-factor refinement with bulksolvent correction and anisotropic B-factor scaling, reduced the R-factor to 0.285 (Rfree = 0.392). The model was fit to the Native 1 datausing rigid-body refinement. Further refinement gave a Rworking of0.268 (Rfree = 0.301) to 1.63 Å resolution with good stereochemistry.The 2|Fo| - |Fc| electron density map calculated at this stage was ofgood quality which allowed errors in the initial model to be correct-ed and missing residues to be built. Cycles of refinement, modelrebuilding and addition of solvent molecules resulted in the currentmodel of the native CAS1 structure with an R-factor of 0.180 (Rfree =0.228) to 1.63 Å resolution. Similar protocols were used for therefinement of the three structures of CAS complexes, except thatthe CAS–Fe(II)–2OG structure was further refined by includinghydrogen atoms and anisotropic displacement with SHELX9746.Electron density for residues 208–214 in CAS–Fe(II)–2OG–NAA and207–214 in CAS–Fe(II)–2OG–A5 was disordered; and these residueswere not included in these models.

Coordinates. Coordinates and the structure factors haven beendeposited in the Protein Data Band (PDB accession codes: NativeCAS: 1DS0, CAS1-Fe(II)-2OG: 1DS1, CAS1-Fe(II)-2OG-NAA: 1DRY,CAS1-Fe(II)-2OG-A5: 1DRT.

Acknowledgments We thank H. McNaughton for purification of proclavaminic acid, M. Groves and I. Clifton for help with computing, S. Lee for photography, D. I Stuart forencouragement, the staff at SRS Daresbury for technical support, our colleagues forencouragement, and the EC, BBSRC, EPSRC and the MRC for financial support.

Correspondence should be addressed to C.J.S. email: [email protected] or K.H. email: [email protected]

Received 15 October, 1999; accepted 23 December, 1999.



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letters

1. Abraham, E. P. & Chain, E. Nature 146, 837 (1940).2. Kirby, W. M. M. Science 99, 452–453 (1944).3. Maiti, S.N., Phillips O.A., Micetich R.G. & Livermore D.M. Current Medicinal

Chemistry. 5, 441–456 (1998).4. Matagne A., Dubus A., Galleni M, Frere J. M. Nat. Prod. Reps. 16, 1–19 (1999).5. Brown, A. G. et al. J. Antibiot. 29, 668–669 (1976).6. Howarth, T. T., Brown, A. G. & King, T. J. J. Chem. Soc. Chem. Commun. 7, 266

(1976).7. Baggaley, K. H., Brown, A. & Schofield, C. J. Nat. Prod. Reps., 14, 309–333 (1997)

and references therein.8. Knowles, J. R. Acc. Chem. Res. 18, 97–104 (1985).9. Brown, R. P., Aplin, R. T. & Schofield, C. J. Biochemistry 35, 12421 (1996)

10. Bentley, P. H. et al. J. Chem. Soc., Chem. Commun. 21, 748–749 (1977).11. Bentley, P. H., Brooks, G., Brooks, G. & Hunt, E. Tetrahedron Lett. 20, 1889–1890

(1979).12. Roach, P. L. et al. Nature 375, 700–704 (1995).13. Roach, P. L. et al. Nature 387, 827–830 (1997).14. Schofield, C. J. et al. Curr. Opin. Struct. Biol. 7, 857–864 (1997).15. Prescott, A. G. J. Exp. Botany 44, 849–861 (1993).16. Valegård, K. et al. Nature 394, 805–809 (1998).17. Lloyd et al., J. Mol. Biol. 287, 943–960 (1999).18. Backmann, B. O., Li, R. & Townsend, C. A. Proc. Natl. Acad. Sci. USA 95, 9082–9086

(1998).19. McNaughton, H. et al. J. Chem. Soc. Chem. Commun. 21, 2325–2326 (1998).20. Nicholson, N. H. et al. J. Chem. Soc. Chem. Commun. 11, 1281–1282 (1994).21. Elson, S.W. et al. J. Chem. Soc. Chem. Commun. 22, 1736–1738 (1987).22. Baldwin, J.E. et al. Tetrahedron 47, 4089–4100 (1991).23. Baldwin, J.E. et al. J. Chem. Soc. Chem. Commun. 6, 500–502 (1993).24. Salowe, S. P., Marsh, E. N. & Townsend, C. A. Biochemistry 29, 6499–6508 (1990).25. Elson, S. W. et al. J. Chem .Soc. Chem. Commun. 15, 1211–1212 (1993).26. Marsh, E.N., Chang, M. D. T. & Townsend, C.A. Biochemistry 31, 12648–12657


(1992).27. Retey, J. Ang. Chem. Intl. Edn. 29, 355–361 (1990).28. Myllyharju, J. & Kivirikko, K.I. EMBO J. 16, 1173–118 (1997).29. Hegg, E. L. & Que, L. Jr. Eur. J. Biochem. 250, 625–629 (1997).30. Holme, E. Biochemistry 14, 4999–5003 (1975).31. Hanauskeabel, H.M., Gunzler, V. J. Theoret. Biol. 94, 21–455 (1982).32. Lange, S.J. & Que, L. Curr. Opin. Chem. Biol. 2, 159–172 (1998).33. Zhou, J., Gunsior, M., Bachmann, B.O., Townsend, C.A.& Solomon, E. I. J. Amer.

Chem. Soc. 120, 13539–13540 (1998).34. Pavel, E. G. et al. J. Amer. Chem. Soc. 120, 743–753 (1998).35. Lloyd, M.D. et al. Tetrahedron 55, 10201–10220 (1999).36. Baldwin, J.E., Adlington R.M., Crouch, N.P.& Pereira, I.A.C. Tetrahedron 49,

7499–7518 (1993).37. Baldwin, J.E. et al. J. Chem. Soc., Chem. Commun. 22, 1694–1696 (1993).38. Baldwin, J.E. et al. Tetrahedron 53, 7011–7020 (1997).39 Lawlor, E.J. et al. Tetrahedron 50, 8737–8748 (1994).40. Zhang, Z.–H., Barlow, J. N., Baldwin, J. E. & Schofield, C. J. Biochemistry 36,

15999–16007 (1997).41. Otwinowski, Z. & Minor, W. Methods Enzymol. 276, 307–326 (1996).42. Otwinowski, Z. In Isomorphous replacement and anomalous scattering (eds

Wolfs, W., Evans, P. R. and Leslie, A. G. W.) 80–85 (SERC Daresbury Laboratory,Warrington, 1991).

43. LaFortelle, E. de and Bricogne, G. Methods Enzymol., 276 472–494 (1996).44. Cowtan, K. In Joint CCP4 and ESF–EACB M Newsletter on protein crystallograhy

34–38 (1994).45. Brunger, A. T. et al. Acta Crystallogr. D 54, 905–921 (1998).46. Sheldrick, G. M. and Schneider, T. R. Methods Enzymol. 277, 319–343 (1997).47. Blundell, T. L. Nature. Struct. Biol. 1, 73–75 (1994).48. Kraulis, P. J. J. Appl. Crystallogr. 24, 946–950 (1991).49. Esnouf, R. M. Acta Crystallogr. D 55, 938–940 (1999).50. Merritt, E. A. & Murphy, M. E. P. Acta Crystallogr. D 50, 869–873 (1994).


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